Impact of porcine circovirus type 2 on porcine epidemic diarrhea virus replication in the IPI-FX cell line depends on the order of infection

Abstract

Introduction: A study in 2006 showed that the clinical course of PEDV disease was markedly aggravated by transplacental infection of PCV2. Therefore, we investigated whether the small intestine supports PCV2 replication and the effect of PCV2 infection on PEDV replication in epithelial cells in vitro.

Methods: To confirm the intestinal tropism of PCV2, the viral loads in the small-intestinal tissues after PCV2 infection were determined with virus titration, and the viral titers in the infected pig jejunum, ileum, ileocecal valve, and colon were 10^4.86, 10^4.09, 10^2.52, and 10^2.35 TCID₅₀/g, respectively. We then determined the propagation characteristics of PCV2 in ileal epithelial cells (IPI-FX) and jejunal epithelial cells (IPEC-J2) with an immunoperoxidase monolayer assay, virus titration, and an immunofluorescence assay. Both IPI-FX and IPEC-J2 cells supported the replication of PCV2, with titers of 10^5.5 and 10^5.0 TCID₅₀/ml, respectively. We established an infection model of PCV2 and PEDV in IPI-FX cells and found that PEDV and PCV2 infected the cells individually and together. The effects of PCV2 infection on PEDV replication were determined with reverse transcription-quantitative PCR (qPCR), western blotting, and virus titration. When PCV2 infected IPI-FX cells before PEDV, PCV2 significantly inhibited the replication of PEDV in a dose- and time-dependent manner and that the mRNAs of IFN-β, TNF-α, IL1β, and OASL were downregulated (detected with qPCR). Surprisingly, when IPI-FX cells were co-infected with PCV2 and PEDV, PCV2 promoted the replication of PEDV, the expression of the host IFN-β, TNF-α, IL1β, and OASL mRNAs was upregulated.

Discussion: These findings demonstrate that the co-infection of IPI-FX cells with PCV2 and PEDV represents an excellent in vitro model in which to investigate their combined pathogenic mechanisms.

Keywords: IPEC-J2 cells; IPI-FX cells; co-infection; porcine circovirus type 2; porcine epidemic diarrhea virus.

Figure 1

Distribution of PCV2 in different segments of porcine intestinal tissues. Three piglets in each group were infected with PCV2d-LNHC or medium (negative control). Samples from different intestinal segments from the six piglets were collected to detect the distribution of PCV2 at 28 dpi. (A) Virus titration was used to determine the titer of PCV2 in the intestinal tissues. The dotted line shows the detection limit, and the value under the dotted line is considered to be ‘undetected’. (B) Number of PCV2 genomes determined with qPCR.

Figure 2

PCV2 replicates in IPI-FX and IPEC-J2 cells. IPI-FX and IPEC-J2 cells were inoculated with PCV2d-LNHC (MOI = 2). After culture for 72 h, the cells were fixed and stained with an IPMA (A). IPI-FX and IPEC-J2 cells were inoculated with PCV2d-LNHC (MOI = 5) and then harvested and freeze–thawed at the indicated times to obtain viral stocks and the viral titers were determined. Growth curves were plotted with the viral titers thus determined (B). IPI-FX cells were inoculated with PCV2d-LNHC. The cells were then fixed and stained with antibodies at the indicated time points, and immunofluorescence was observed with laser confocal microscopy (C).

Figure 3

IPI-FX cells support the replication of both PEDV and PCV2 together. IPI-FX cells were inoculated with both PEDV and PCV2d-LNHC. At 72 h after inoculation, the cells were fixed, stained with an immunofluorescence assay (IFA), and observed with laser confocal microscopy. White arrows indicate cells infected with PEDV and PCV2.

Figure 4

PCV2 reduces PEDV replication in IPI-FX cells. IPI-FX cells were inoculated PCV2d-LNHC at MOI = 2, 1, 0.5, 0.25, or 0.1 and incubated for 24 h. The same cells were then inoculated with PEDV (MOI = 2 or 5) and incubated for another 24 h. The cells were collected, the total RNA extracted, and the relative expression levels of nucleocapsid (N) mRNA in the PEDV–PCV2 dual-infected or PEDV-infected cells determined with RT–qPCR (A,B). IPI-FX cells were inoculated PCV2a-LG or PCV2b-MDJ at MOI = 2, 1, 0.5, 0.25, or 0.1 and incubated for 24 h. The same cells were then inoculated with PEDV (MOI = 5) and incubated for another 24 h. The cells were collected, the total RNA extracted, and the relative expression levels of nucleocapsid (N) mRNA in the PEDV–PCV2 dual-infected or PEDV-infected cells determined with RT–qPCR (C,D). IPI-FX cells were inoculated PCV2d-LNHC at MOI = 2, 1, 0.5, 0.25, or 0.1 and incubated for 24 h. The same cells were then inoculated with PEDV (MOI = 5) and incubated for another 24 h. After that, the cells were collected, lysed, and analyzed with western blotting using the antibodies indicated on the left. GADPH was loaded as the internal control (E). IPI-FX cells were inoculated PCV2d-LNHC at MOI = 2, 1, 0.5, 0.25, or 0.1 and incubated for 24 h. The same cells were then inoculated with PEDV (MOI = 5) and incubated for another 24 h. The cells and supernatants were freeze–thawed to obtain viral stocks. The viral titers were determined with the Reed–Muench method (F). IPI-FX cells were inoculated with PCV2d-LNHC (MOI = 2 or 1) and incubated for 24 h with or without d-glucosamine. The cells were then inoculated with PEDV (MOI = 5) and incubated for another 24 h. The cells were collected, the total RNA extracted, and the relative expression levels of nucleocapsid (N) mRNA in the PEDV–PCV2 dual-infected or PEDV-infected cells determined with RT–qPCR (G). IPI-FX cells were inoculated with PCV2d-LNHC (MOI = 2) and incubated for the indicated times, and then with PEDV (MOI = 5) and incubated for another 24 h. The cells were collected, the total RNA extracted, and the relative expression levels of nucleocapsid (N) mRNA in the PEDV–PCV2 dual-infected or PEDV-infected cells determined with RT–qPCR (H). IPI-FX cells were inoculated with PCV2d-LNHC (MOI = 2) and incubated for 24 h, and then with PEDV (MOI = 5) and incubated for another 24 h. The cells were collected, the total RNA extracted, and the relative expression levels of IL1β, IFN-β, OASL, and TNF-α mRNAs in the PEDV–PCV2 dual-infected and PEDV-infected cells determined with RT–qPCR using specific primers for IL1β, IFN-β, OASL, and TNF-α (I). GADPH mRNA was used as the internal control for normalization (A–I). Means ± standard deviations were calculated from three independent replicates of each experiments.

Figure 5

PCV2 promotes PEDV replication in IPI-FX cells. IPI-FX cells were inoculated with PEDV (MOI = 2 or 5) and simultaneously with PCV2d-LNHC (MOI = 1, 0.5, or 0.25). The cells were collected, the total RNA extracted, and the relative expression levels of nucleocapsid (N) mRNA in the PEDV–PCV2 simultaneously infected or PEDV-infected cells determined with RT–qPCR at 24 hpi (A,B). IPI-FX cells were inoculated with PEDV (MOI = 5) and simultaneously with PCV2a-LG or PCV2b-MDJ (MOI = 1, 0.5, or 0.25). The cells were collected, the total RNA extracted, and the relative expression levels of nucleocapsid (N) mRNA in the PEDV–PCV2 simultaneously infected or PEDV-infected cells determined with RT–qPCR at 24 hpi (C,D). IPI-FX cells were inoculated with PEDV (MOI = 5) and simultaneously with PCV2d-LNHC (MOI = 1, 0.5, or 0.25). At 24 hpi, the cells were collected, lysed, and analyzed with western blotting using the antibodies indicated on the left. GADPH was loaded as the internal control (E). Western blotting results were quantified with ImageJ (National Institutes of Health, Bethesda, Maryland, United States) (F). IPI-FX cells were inoculated with PEDV (MOI = 5) and simultaneously with PCV2d-LNHC (MOI = 1, 0.5, or 0.25). At 24 hpi, the cells and supernatants were freeze–thawed to obtain viral stocks. The viral titers were determined with the Reed–Muench method (G). Relative expression levels of IL1β, IFN-β, OASL, and TNF-α mRNAs in the PEDV–PCV2 simultaneously infected and PEDV-infected cells were also determined (H). GADPH mRNA was used as the internal control for normalization. Means ± standard deviations were calculated from three independent replicates of each experiment (A–H).

Figure 6

PCV2 tolerates environmental pH 2.0. PK15 cells were inoculated with PCV2d-LNHC at pH 2.0 and incubated for 1–4 h at 37°C. The viral titers in the acid-treated PCV2-infected and mock-PCV2-infected cells were determined with virus titration.